Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
Cell Mol Life Sci. 2018 Oct;75(19):3635-3647. doi: 10.1007/s00018-018-2821-0. Epub 2018 Apr 21.
When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up to ~ 65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze-thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica. Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.
当温度骤降至 0°C 以下时，林蛙（Rana sylvatica）可以忍受其多达约 65%的细胞外冰体内水分的冻结，此时没有可测量的大脑活动、呼吸、运动，并且心脏呈直线状。为了帮助生存，青蛙进入休眠状态，其代谢功能全面受到抑制，能量使用重新分配到对基本生存过程的支持，这部分是由 microRNAs 的调控控制引起的。本研究首次调查了耐寒脊椎动物大脑中的 miRNA 生物发生和调控。事实上，大脑的正常功能和对环境刺激的适应在协调应激反应方面起着至关重要的作用。miRNA 生物发生因子的免疫印迹显示，这些加工蛋白的大多数总体减少，这表明在冷冻-解冻循环中可能抑制了 miRNA 的成熟。这与对对照、24 小时冷冻和 8 小时解冻的 R. sylvatica 大脑中的 113 种 microRNA 物种的相对表达水平进行的大规模 RT-qPCR 分析相结合。在冷冻和解冻过程中差异表达的 41 个 microRNAs 中，只有两个明显上调。在冷冻和解冻过程中经历的低温下对下调的 microRNAs 进行的生物信息学靶标富集预测了它们参与潜在激活各种神经保护过程的作用，如突触信号、细胞内信号转导和缺氧/缺血损伤保护。本文中确定的主要下调的 microRNA 指纹表明存在一种 microRNA 介导的冷冻保护机制，负责维持神经元功能并促进整个大脑的成功冷冻和解冻。
